«Kinetic Investigations of Thiolate Protected Gold Nanoparticles: Protein Interactions, Electron Transfer, and Precursor Formation By Brian N. Turner ...»
Following the place exchange reaction, the material consists of unbound thiols from both the incoming and outgoing ligands, consequently requiring an additional purification step. Removal of these impurities is a unique challenge as the different molecules not only have different preferences for solvents (in the case of classical wet chemical purification techniques) or pore sizes (in the case of a size-exclusion technique), but properties that are similar to the nanoparticle. Options that are explored in subsequent chapters are washing with a battery of solvents, solvent immersion and sonication on glass, suspension and centrifugation given the availability of a sufficient solvent for this purpose, suspension and precipitation from cold solvent, dialysis, and molecular weight cut-off centrifuge filters. Some other suggestions for purification are continuous freeflow electrophoresis,54 and diafiltration.55 Besides the place-exchange strategy, there are other strategies to functionalize MPCs. An important class of these strategies, which is beginning to gain popularity, is the use of simple organic reactions on ligands already bound to the MPC. Examples include triazole cycloaddition to a bromine functionality56 and direct functionalization of a hydroxyl group,57 amide coupling and ester coupling.58 All of these methods allow for post-exchange reaction chemistry to occur, yielding surfaces that can be modified in a controlled fashion.
Another popular option, not explored in this dissertation, is polyfunctionalization in situ.
When the properties of the given ligands will allow it, a mixed ligand nanoparticle synthesis is also an option. Mixed ligand synthesis refers to a reaction where a certain percentage mixture of two or more thiolated ligands are added to the gold salt prior to reduction, resulting in polyfunctionalized nanoparticle similar to those produced via place-exchange. Mixed ligand synthesis has the advantage of not requiring additional mixing and purification steps, but it is more difficult to control the pre-reduction chemistry and therefore more difficult to control the extent of exchange.
Characterization Methods for MPCs A thorough characterization of mono- and polyfunctionalized nanoparticles is critical before applying them in biological or materials uses. Determination of core size (greater than about 1 nm, depending upon the substrate used) and dispersity is easily accomplished via transmission electron microscopy (TEM). 4 Core sizes have also been determined by mass spectrometric methods, but this is limited to smaller cores with charge carrying ligands.59, 60 Scattering techniques and zeta-potential instruments are not as helpful for this class of nanomaterial due to the complexities that come with combined absorbance and scattering. Thermogravimetric analysis (TGA) 61 provides an easy method to determine the ratio of organic ligand to inorganic core material. Combining the core size and organic/inorganic ratio data yields an approximate average molecular formula for homofunctionalized nanoparticles. 60 Nuclear magnetic resonance spectroscopy (NMR) is useful for determining the structure and composition of the protecting monolayer, as well as the purity of the final material. Protecting ligands have broadened peaks in both 1H and 13C spectra due to spin-spin relaxational (T2) broadening, dispersity in binding sites, and dipolar broadening due to packing density gradients. 62, 63 Once functionalized into a useful conjugate material, it is critical to characterize MPCs in terms of the quantity of biological relevant functional groups attached per cluster (on average) and the secondary structure of the biomimics post-conjugation. 1H NMR is a simple way to semi-quantitatively determine the number of antigen peptides per cluster via integration of known protecting ligand peaks versus new broadened biomolecule peaks. The accuracy of this method can be enhanced through the use of I 2-induced MPC decomposition (termed the “death reaction”) which leads to sharper peaks with less overlap.64 A newer technique that has been developed in our group for relative surface quantitation of polyfunctionalized nanoparticles is matrix assisted laser desorption ionization-ion mobility-mass spectrometry (MALDI-IM-MS).65 This specific mass spectrometric technique has the advantage of sorting out impurities with small collisional cross sections from the gold complexes actually used to quantify the surface (see the methods section of Chapter IV for more information). Secondary structure determination has proven to be more difficult. Drobny and co-workers describe the use of novel solidstate NMR techniques to investigate the secondary structure of peptides immobilized on gold MPCs via amide coupling.66 In an elegant study, they were able to use crosspolarization magic angle spinning (CPMAS) with double-quantum dipolar recoupling with a windowless sequence (DQDRAWS) to show that a peptide maintained a helical structure upon conjugation, but with a slight change in backbone torsion angle. Mandal and Kraatz recently described similar characterizations of peptides place-exchanged onto MPCs using Fourier transform infrared spectroscopy (FT-IR) and Fourier transform reflection absorption spectroscopy (FT-RAIRS).67 Using amide I bands, they were able to observe that the secondary structure of a leucine rich peptide bound to gold transitions from α-helical to β-sheet with greater surface curvature. Helix forming free peptides, 2D SAMs on gold, and peptides on 20 nm gold MPCs (less curvature) showed α-helical structure while 10 nm, and especially 5 nm gold MPCs (more curvature) showed increasing amounts of β-sheet conformation. An excellent review of characterization techniques for peptide-nanoparticle conjugates was written by Slocik and Naik. 68 In this review, they describe a number of methods based on NMR, CD, FT-IR, and binding assays to reveal or confirm secondary structure of the conjugated peptides. Consideration of these techniques should prove vital in improving the characterization quality of biomolecule conjuagated nanomaterials in our research group.
Instrumentation for Routine Nanoparticle Characterization Nanoparticle characterization was accomplished with a variety of instrumentation. 1H NMR spectra for synthetic characterization and purity confirmation were collected using a Bruker AV 400 MHz NMR. Surface ligand quantitation was accomplished with Bruker DRX 500 MHz, and AV 600 MHz instruments. Ultraviolet/Visible spectroscopy was performed using a Cary 100 Bio UV/Visible spectrophotometer. Thermogravimetric analysis (TGA) was performed in the Vanderbilt Institute of Nanoscale Science and Engineering (VINSE) Biomolecular Nano-Structure lab using an Instrument Specialist’s TGA 1000 with N2 or air as the chamber gas. Transmission electron microscopy was performed using a Philips CM20 TEM with a LaB 6 tip operating at 200 kV. Samples are prepared by a variety of methods on formvar or ultrathin holey carbon supported on copper mesh grids, purchased from Ted Pella.
LINEAR EPITOPE MAPPING APPROACH TO LOCATE BINDING “HOT SPOTS”
IN THE CONFORMATIONAL ANTIGENIC SITE A OF HUMAN RESPIRATORY
SYNCYTIAL VIRUS (HRSV) FUSION PROTEIN (F)
Having discussed the synthesis and characterization of thiolate protected nanoparticle synthesis, the application of these materials in a biological context will be discussed.
The question, “How can you make a small gold nanoparticle resemble a protein in a way that it “tricks” a system into thinking that it really is one?” will be asked. Some answers to this question will be covered in the next two chapters. Chapter II deals with a biological system that is mimicked, and the results of that study are important to the construction of a bio-nano-conjugate material, discussed in Chapter III. Specifically, the research aimed to determine which piece of a full protein is necessary to achieve a specific function. In the current case, it was desirable to know which peptide segment from HRSV F protein is necessary for recognition of a specific antibody and whether it still behaves in the same way when not part of the native protein. Furthermore, the impact of removing or adding amino acids in a stepwise fashion on the strength of the recognition was studied. The effect of removing and adding terminal amino acids was initially probed using the quartz crystal microbalance, and later with enzyme-linked immunosorbant assay (ELISA).
It is important to note that, ultimately, this research came together in a non-linear fashion.
Summarily, preliminary experiments with the QCM to examine the antibody binding properties of relevant peptides were attempted with little success, prompting the research to pursue peptide conjugated nanoparticles not informed by epitope mapping experiments. Having not found a strong, specific nanoparticle biomimic in the first round of experiments, epitope mapping was revisited with ELISA. The second round of epitope mapping resulted in more informative data, presented in this chapter, which allowed for construction of an improved nanoparticle mimic. The improvements and results to the nanoparticle mimic are presented in Chapter III. The results were then put back together in a more readable account of the research than if they were presented chronologically.
Therefore, while experiments from Chapter II and III alternate in time and inform each other, they are more cohesive in the current structure.
Human Respiratory Syncytial Virus Acute respiratory tract infection is the leading cause of death by infectious disease worldwide and HRSV is the leading cause of acute respiratory tract infection in infants. 69 A 13-year surveillance of Washington DC infants and young children showed that HRSV was present in 43% of patients with bronchiolitis and 25% of patients with pneumonia. 70 Diagnosis of HRSV can be difficult during the cold season due to an increased patient load, especially given the high volume of young patients with associated symptoms from other infections. The insufficient pace of clinical diagnosis often results in longer than necessary hospital stays for afflicted children and their families, and an increased financial burden for hospitals. Our research group is focused on strategies to develop quick and accurate diagnostic platforms, to analyze immunological interactions of HRSV with the commercial pharmaceutical IgG Palivizumab (PZ), and to design biological mimics of this immunological interaction.
The HRSV virion is in the family Paramyxoviridae and of the order Mononegavirales, making it a non-segmented negative sense RNA virus.69 HRSV is in the subfamily Pneumovirinae along with bovine respiratory syncytial virus (BRSV), pneumonia virus of mice (PVM), and avian and human metapneumovirus (AMPV and HMPV). 69 The diversity in this family of viruses allows researchers to tune strategies developed for one virus to a number of different diseases because of structural similarities.
A general understanding of the protein structure and function is vital to understanding the goals and approaches of this project. A structural schematic of HRSV is shown in Figure 7.
Figure 7: Cartoon schematic of an HRSV virion. The RNA and associated proteins are housed by a matrix protein which is covered with a protective lipid bilayer. The lipid bilayer projects three different surface glycoproteins that are important targets for the immune system: fusion (F), attachment (G), and small hydrophobic (SH). Figure from the Katholieke Universiteit Leuven, used with permission. 71 The viral genome encodes three surface glycoproteins (fusion [F], attachment [G], and a small hydrophobic protein [SH]). Also encoded are a non-glycosylated matrix protein (M), a nucleoprotein (N), a phosphoprotein (P), transcription processivity factors, two non-structural proteins, and a large viral RNA polymerase unit (L). The structure of the virion houses a nucleoprotein/RNA complex payload at its core (L, N, P and possibly transcription processivity factors).69 The RNA is thought to be surrounded by a protective coat of M. HRSV is an enveloped virus; therefore it collects a lipid bi-layer upon budding from infected cells, making it more difficult for the immune system to recognize and destroy. The three surface glycoproteins project from the surface of this envelope as 11-20 nm spikes spaced at about 6-10 nm apart.69 The total size of the virus varies from 100-350 nm diameter spherical particles to 10 m long filamentous particles having a radius of 60-200 nm.69 Analysis of the proteins encoded for by HRSV RNA has allowed researchers to identify appropriate targets for detection and treatment strategies.
Of all the encoded HRSV proteins, F and G are the only two that induce neutralizing antibodies, as steric hindrance at certain areas of these sites will block cell entry. 72 The importance of the third surface glycoprotein (SH) is not clear, and it has been shown to be non-essential both in vitro and in vivo.73, 74 G is important for viral attachment and F accomplishes viral fusion; therefore, if an antibody binds either one of these antigens at an epitope that is simultaneously involved in a critical function, viral replication will be neutralized by sterics and the prevention of necessary conformational changes.
Consequently, a wide variety of neutralizing monoclonal antibodies are known for each.
G, which is heavily glycosylated, has a highly variable amino acid sequence from strain to strain, whereas neutralizing antibodies targeted against F are cross reactive between various strains and across antigenic subgroups.75 These properties render F a superior target for antibody-based detection strategies.
In the absence of an available crystal structure of HRSV F protein, the best resource to gain structural insights to aid this project is a Newcastle Disease Virus (NDV) fusion protein homology model of HRSV F.76, 77 A structural schematic of the protein illustrates the trimeric structure, and presence of stalk, neck, and head regions (see Figure 8).
Figure 8: HRSV F protein, showing the head, neck, and stalk structure. The head docks with the cell wall during infection, and is the site of epitopes for neutralizing mAbs.
Figure from Morton, et. al.76 Copyright 2003, Elsevier, used with permission.